780 meta

Immunofluorescence was performed by using previously published methods [45 (link)]. Briefly, oocytes were fixed for 30 min at room temperature in 2% formaldehyde supplemented with 100mM HEPES, 50mM EGTA, 10mM MgSO₄, 0.2% Triton X-100 (pH=7, titrated with KOH). Then they were treated with PBS, 0.1% Triton X-100 overnight at 4^oC and incubated with anti-α-tubulin-FITC antibody (F2168, Sigma, USA) (1:1000 in PBS with 0.1% Triton X-100 and 3% BSA) overnight at 4^oC. Chromosomes were stained with DAPI for 15 min. The oocytes were mounted on glass slides and examined with a laser scanning confocal microscope (Zeiss 780 META).
For staining active mitochondria, eggs were incubated for 30 min at 37°C in M2 medium containing 200nM MitoTracker ® Red CMXRos (M7512, Invitrogen, USA). After washing 3 times, eggs were stained with Hoechst 33342 (10mg/ml) for 15 min. Finally, eggs were mounted on glass slides and examined with a Perkin Elmer UltraVIEW VOX confocal Imaging System.

Ovaries used for immunostaining were fixed in 4% paraformaldehyde (pH 7.4) overnight at 4 ℃, dehydrated, and embedded in paraffin. Paraffin-embedded ovaries were cut into sections of 5-μm thickness. Then, the sections were deparaffinized, immersed in sodium citrate buffer (pH 6.0), and heated for 15 min in a microwave for antigen retrieval. After blocking with 5% donkey serum albumin, sections were incubated with primary antibodies at 4 °C overnight. For immunohistochemistry, the sections were treated with 3% H₂O₂ to eliminate internal peroxidase activity and incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Finally, the signal of primary antibody was detected by the Vectastain ABC kit (Vector Laboratories, CA, USA) and the sections were counterstained with hematoxylin. Images were captured using a Nikon DS-Ri1 CCD camera. For immunofluorescence, the sections were incubated with an appropriate FITC-conjugated secondary antibody. The nuclei were stained with DAPI. Images were captured using a laser scanning confocal microscope (Zeiss 780 META).

Testes or caudal epididymides were dissected from Ck2β^fl/Δ and Ck2β^fl/Δ;SCre⁺ male mice immediately after euthanasia. Testes were fixed in 4% paraformaldehyde (pH 7.4) or Bouin's fixative overnight at 4℃. Caudal epididymides were fixed in 4% paraformaldehyde (pH 7.4) overnight at 4℃. The tissues were then dehydrated in a graded ethanol series, cleaned in xylene and embedded in paraffin. The paraffin‐embedded tissues were sectioned into 5 μm and mounted on glass slides. After adequately drying at 48℃, the sections were deparaffinized in xylene, hydrated in a graded ethanol series and stained with haematoxylin and eosin for histological analysis.
Testes for immunofluorescent staining were fixed in 4% paraformaldehyde (pH 7.4) overnight at 4℃, dehydrated and embedded in paraffin. The paraffin‐embedded testes were sectioned into 5 μm and mounted on glass slides. The sections were then deparaffinized in xylene, hydrated in a graded ethanol series, immersed in sodium citrate buffer (pH 6.0) and heated for 15 minutes in a microwave oven for antigen retrieval. After blocking with 5% donkey serum albumin, the sections were incubated with primary antibody at 4℃ overnight and appropriate TRITC‐conjugated secondary antibody. The nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI). Images were captured using a laser scanning confocal microscope (Zeiss 780 META).